MAGE-10 encoding cDNA, the tumor rejection antigen precursor MAGE-10, antibodies specific to the molecule, and uses thereof

ABSTRACT

Isolated cDNA molecules which encode the tumor rejection antigen precursor MAGE-10, the protein itself, antibodies to it, and uses of these are a part of the invention.1

FIELD OF THE INVENTION

[0001] This invention relates to tumor rejection antigen precursors, thenucleic acid molecules encoding them, antibodies specific to these, anduses thereof.

BACKGROUND AND PRIOR ART

[0002] The study of the recognition or lack of recognition of cancercells by a host organism has proceeded in many different directions.Understanding of the field presumes some understanding of both basicimmunology and oncology.

[0003] Early research on mouse tumors revealed that these displayedmolecules which led to rejection of tumor cells when transplanted intosyngeneic animals. These molecules are “recognized” by T-cells in therecipient animal, and provoke a cytolytic T-cell response with lysis ofthe transplanted cells. This evidence was first obtained with tumorsinduced in vitro by chemical carcinogens, such as methylcholanthrene.The antigens expressed by the tumors and which elicited the T-cellresponse were found to be different for each tumor. See Prehn, et al.,J. Natl. Canc. Inst. 18: 769-778 (1957); Klein et al., Cancer Res. 20:1561-1572 (1960); Gross, Cancer Res. 3: 326-333 (1943), Basombrio,Cancer Res. 30: 2458-2462 (1970) for general teachings on inducingtumors with chemical carcinogens and differences in cell surfaceantigens. This class of antigens has come to be known as “tumor specifictransplantation antigens” or “TSTAs”. Following the observation of thepresentation of such antigens when induced by chemical carcinogens,similar results were obtained when tumors were induced in vitro viaultraviolet radiation. See Kripke, J. Natl. Canc. Inst. 53: 333-1336(1974).

[0004] While T-cell mediated immune responses were observed for thetypes of tumor described supra, spontaneous tumors were thought to begenerally non-immunogenic. These were therefore believed not to presentantigens which provoked a response to the tumor in the tumor carryingsubject. See Hewitt, et al., Brit. J. Cancer 33: 241-259 (1976).

[0005] The family of tum⁻ antigen presenting cell lines are immunogenicvariants obtained by mutagenesis of mouse tumor cells or cell lines, asdescribed by Boon et al., J. Exp. Med. 152: 1184-1193 (1980), thedisclosure of which is incorporated by reference. To elaborate, tum⁻antigens are obtained by mutating tumor cells which do not generate animmune response in syngeneic mice and will form tumors (i.e., “tum⁺”cells). When these tum⁺ cells are mutagenized, they are rejected bysyngeneic mice, and fail to form tumors (thus “tum⁻”). See Boon et al.,Proc. Natl. Acad. Sci. USA 74: 272 (1977), the disclosure of which isincorporated by reference. Many tumor types have been shown to exhibitthis phenomenon. See, e.g., Frost et al., Cancer Res. 43: 125 (1983).

[0006] It appears that tum⁻ variants fail to form progressive tumorsbecause they initiate an immune rejection process. The evidence in favorof this hypothesis includes the ability of “tum⁻” variants of tumors,i.e., those which do not normally form tumors, to do so in mice withimmune systems suppressed by sublethal irradiation, Van Pel et al.,Proc. Natl. Acad. Sci. USA 76: 5282-5285 (1979); and the observationthat intraperitoneally injected tum⁻ cells of mastocytoma P815 multiplyexponentially for 12-15 days, and then are eliminated in only a few daysin the midst of an influx of lymphocytes and macrophages (Uyttenhove etal., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes theobservation that mice acquire an immune memory which permits them toresist subsequent challenge to the same tum⁻ variant, even whenimmunosuppressive amounts of radiation are administered with thefollowing challenge of cells (Boon et al., Proc. Natl, Acad. Sci. USA74: 272-275 (1977); Van Pel et al., supra; Uyttenhove et al., supra).Later research found that when spontaneous tumors were subjected tomutagenesis, immunogenic variants were produced which did generate aresponse. Indeed, these variants were able to elicit an immuneprotective response against the original tumor. See Van Pel et al., J.Exp. Med. 157: 1992-2001 (1983). Thus, it has been shown that it ispossible to elicit presentation of a so-called “tumor rejection antigen”in a tumor which is a target for a syngeneic rejection response. Similarresults have been obtained when foreign genes have been transfected intospontaneous tumors. See Fearon et al., Cancer Res. 48: 2975-1980 (1988)in this regard.

[0007] A class of antigens has been recognized which are presented onthe surface of tumor cells and are recognized by cytolytic T cells,leading to lysis. This class of antigens will be referred to as “tumorrejection antigens” or “TRAs” hereafter. TRAs may or may not elicitantibody responses. The extent to which these antigens have beenstudied, has been via cytolytic T cell characterization studies, invitro i.e., the study of the identification of the antigen by aparticular cytolytic T cell (“CTL” hereafter) subset. The subsetproliferates upon recognition of the presented tumor rejection antigen,and the cells presenting the tumor rejection antigens are lysed.Characterization studies have identified CTL clones which specificallylyse cells expressing the tumor rejection antigens. Examples of thiswork may be found in Levy et al., Adv. Cancer Res. 24: 1-59 (1977); Boonet al., J. Exp. Med. 152: 1184-1193 (1980); Brunner et al., J. Immunol.124: 1627-1634 (1980); Maryanski et al. , Eur. J. Immunol. 124:1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982);Palladino et al., Canc. Res. 47: 5074-5079 (1987). This type of analysisis required for other types of antigens recognized by CTLs, includingminor histocompatibility antigens, the male specific H-Y antigens, andthe class of antigens referred to as “tum−” antigens, and discussedherein.

[0008] A tumor exemplary of the subject matter described supra is knownas P815. See DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278(1988); Szikora et al., EMBO J 9: 1041-1050 (1990), and Sibille et al.,J. Exp. Med. 172: 35-45 (1990), the disclosures of which areincorporated by reference. The P815 tumor is a mastocytoma, induced in aDBA/2 mouse with methylcholanthrene and cultured as both an in vitrotumor and a cell line. The P815 line has generated many tum⁻ variantsfollowing mutagenesis, including variants referred to as P91A (DePlaen,supra), 35B (Szikora, supra), and P198 (Sibille, supra). In contrast totumor rejection antigens—and this is a key distinction—the tum⁻ antigensare only present after the tumor cells are mutagenized. Tumor rejectionantigens are present on cells of a given tumor without mutagenesis.Hence, with reference to the literature, a cell line can be tum⁺, suchas the line referred to as “P1”, and can be provoked to produce tum⁻variants. Since the tum⁻ phenotype differs from that of the parent cellline, one expects a difference in the DNA of tum⁻ cell lines as comparedto their tum⁺ parental lines, and this difference can be exploited tolocate the gene of interest in tum⁻ cells. As a result, it was foundthat genes of tum⁻ variants such as P91A, 35B and P198 differ from theirnormal alleles by point mutations in the coding regions of the gene. SeeSzikora and Sibille, supra, and Lurquin et al., Cell 58: 293-303 (1989).This has proved not to be the case with the TRAs of this invention.These papers also demonstrated that peptides derived from the tum⁻antigen are presented by the L^(d) molecule for recognition by CTLs.P91A is presented by L^(d), P35 by D^(d) and P198 by K^(d).

[0009] PCT application PCT/US92/04354, filed on May 22, 1992 assigned tothe same assignee as the subject application, teaches a family of humantumor rejection antigen precursor coding genes, referred to as the MAGEfamily. Several of these genes are also discussed in van der Bruggen etal., Science 254: 1643 (1991). It is now clear that the various genes ofthe MAGE family are expressed in tumor cells, and can serve as markersfor the diagnosis of such tumors, as well as for other purposesdiscussed therein. See also Traversari et al., Immunogenetics 35: 145(1992); van der Bruggen et al., Science 254: 1643 (1991) and De Plaen,et al., Immunogenetics 40: 360 (1994).

[0010] U.S. Pat. No. 5,342,774, cited supra and incorporated byreference, teaches various members of the MAGE family of TRAPs, ingenomic DNA and cDNA form. Genomic DNA for MAGE-10 is taught in PCTapplication PCT/US92/04354, cited supra, in SEQ ID NO: 22, as a 920 basepair fragment. DePlaen, et al., Immunogenetics 40: 360-369 (1994),discusses PCR work which identified a 485 nucleotide portion of MAGE-10.Also, see Genbank Accession No. U10685, incorporated by reference. AcDNA molecule, however, is not discussed.

[0011] The previously cited PCT application discusses antibodies to MAGEproteins generally. Chen et al., U.S. Pat. No. 5,541,104, to Chen etal., incorporated by reference, teaches monoclonal antibodies whichspecifically bind to tumor rejection antigen precursor MAGE-1. Thispatent is incorporated by reference. In order to prepare the monoclonalantibodies, Chen et al produced a MAGE-1 TRAP in E. coli which was notfull length, because of difficulties with expression of the full lengthmolecule.

[0012] It has now been found, however, that monoclonal antibodies whichbind to both MAGE-1 and MAGE-10 TRAP can be produced. This is surprisingin view of the reports in the literature, because it was not seen to bepossible to produce such antibodies with the available information onMAGE-10. The TRAP encoded by the cDNA for MAGE-10 is found to be amolecule of about 72 kilodaltons molecular weight, on SDS-PAGE. It hasalso been found that polyclonal antibodies specific to MAGE-10 can beproduced. These, as well as other aspects of the invention, are setforth in the disclosure which follows.

BRIEF DESCRIPTION OF THE DPAWINGS

[0013]FIG. 1 presents results of a Western blotting assay, usingelectrochemiluminescence detection to test reactivity of monoclonalantibodies with various cell lysates.

[0014]FIG. 2 presents results of tests designed to determine if cellline NA8-MEL could be induced to produce 72 kilodalton protein in thepresence of MAGE-1 cDNA.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

[0015] Full length recombinant MAGE-1 protein was prepared in the formof a fusion protein, in E. coli. See Schultz-Thater, et al., Int. J.Cancer 59: 435-439 (1994), incorporated by reference. Briefly, fulllength MAGE-1 cDNA was cloned into a well known expression vector, pET16b. This vector permits expression of a fusion protein which contains10 histidine molecules at the N-terminus. The E. coli were cultured,following Schultz-Thater, after which the cells were lysed, and therecombinant fusion protein was purified on a Ni²⁺ column. The purifiedmaterial, when tested by SDS-PAGE, showed a major band of 48kilodaltons. This 48 kD material was used in the experiments whichfollow.

EXAMPLE 2

[0016] Following the production of the recombinant MAGE-1 fusionprotein, a BALB/c mouse was immunized intraperitoneally, twice, with 20ug of the recombinant protein each time, in a composition whichcontained complete Freund's adjuvant. This was followed by twoadditional injections, each of 20 ug of recombinant MAGE-1, withincomplete Freund's adjuvant. The spleen cells of the mouse were thenfused with NS-1 myeloma cells, in accordance with Carrel, et al., CancerRes. 40: 2523-2528 (1980). The resulting hybridoma cells were cultured,and supernatants from the cultures were screened, using an ELISA, todetermine if recombinant MAGE-1 specific monoclonal antibodies werebeing produced. The ELISA involved coating recombinant MAGE-1 protein(250 ng/50 ul per well), followed by overnight incubation at 4° C.Samples of supernatant were added, followed by biotin conjugated sheepantimouse Ig, and streptavidin-alkaline phosphatase conjugate.

[0017] The ELISA resulted in the identification of 289 hybridomas whichproduced antibodies against recombinant MAGE-1.

EXAMPLE 3

[0018] In the next set of experiments, the antibodies were tested todetermine if they could be used to immunostain cells which were positivefor mRNA for MAGE-1.

[0019] Initially the hybridomas were screened to try to eliminate anycross reactive monoclonals. To do this, cell lines with known, anddifferent patters of MAGE-TRAP expression were tested. MZ2-MEL 3.1 isknown to express all of MAGE-1, 2, 3 and 4; MZ2-MEL 2.2 expresses MAGE-2and 3; and U251, a glioblastoma cell line negative for all four, weretested. Cells were cultured in 16 well plastic chambers, fixed in coldacetone (0° C. for five minutes), and then stored until ready to use at20° C. Endogenous peroxidase was then blocked with 0.3% H₂O₂ (10minutes), and the cells were then preincubated, in 0.1% bovine serumalbumin in phosphate buffered saline, for 30 minutes. This produced afirst layer of a three layer biotin/avidin/peroxidase system asdescribed by Carrel, et al., supra. Following the fixing of the cells,goat anti-mouse IgG biotin conjugate was added (following 1:50dilution), to yield the second layer. Finally, avidin-peroxidaseconjugates were added, following dilution at 1:1000. In the case of thesecond and third layers, incubation was for 30 minutes and then 15minutes. Peroxidase was visualized with amino-ethylcarbazole, andcounter staining of cells, using Gill's hematoxylin for 30 seconds. Thisset of experiments results in the discovery that two mAbs, i.e., 6C1 and6F2, stained only the MZ2-MEL 3.1 cells. These two clones were then usedin a series of experiments on cells which had been tested for mRNA forMAGE-1, 2, 3 and 4. Cells were classified as being positive or negativefor MAGE-1 mRNA expression. This was determined by following theprocedures of Rimoldi et al., Int. J. Cancer 54: 527-528 (1993);Brasseur et al., Int. J. Cancer 63: 375-380 (1995). In brief, total RNAwas extracted from cell samples using well known, commercially availablemethods and reagents, and then subjected to reverse transcription andpolymerase chain reaction using MAGE-1, MAGE-2, MAGE-3 and MAGE-4specific primers. See Brasseur, et al, supra. Table 1, which follows,presents the results of this work. It shows that, regardless of statusof MAGE-2, 3 or 4 expression both mAbs stained all MAGE-1 positivecells. TABLE I IMMUNOCYTOCHEMICAL REACTIVITY

6Cl AND 6F12 WITH VARIOUS CELL LINES Immunostaining¹ MAGE-mRNA Celllines MAb 6Cl MAb 6F12 expression² ^((a))MZ2 · MEL 3.1 + + 1^(⊕), 2^(⊕),3^(⊕), 4^(⊕)

(

)MZ2-MEL 2.2 − − 1−, 2^(⊕), 3^(⊕), 4−

(

)MZ2-MEL 2.2 ET1 + + 1^(⊕), 2^(⊕), 3^(⊕), 4−

(

)Me235 + + 1^(⊕), 2^(⊕), 3^(⊕), 4−

(

)Mi13443 + + 1^(⊕), 2^(⊕), 3^(⊕), 4^(⊕)

(

)NA8-MEL − − 1−, 2−, 3−, 4−

(

)Me220 − − 1−, 2−, 3−, 4−

(

)Me241-2 + + 1^(⊕), 2−, 3−, 4−

(

)Mi9 − − 1−, 2^(⊕), 3^(⊕), 4− ^((a))Mi13 − − 1−, 2^(⊕), 3^(⊕), 4−^((a))Mi21 − − 1−, 2−, 3^(⊕), 4− ^((b))U251 − − 1−, 2−, 3−, 4−^((c))MCF7 − − 1−, 2−, 3−, 4− ^((d))FeK4 − − 1−, 2−, 3−, 4− ^((e))P815 −− 1−, 2−, 3−, 4− ^((f))P815/MAGE-1 + + 1^(⊕), 2−, 3−, 4− ^((g))HEL + +1^(⊕), 2−, 3−, 4− ^((g))TF1 − − 1−, 2−, 3^(⊕), 4−

EXAMPLE 4

[0020] A further set of experiments were then carried out, using thewell known Western blotting technique. Five cell lines were tested,i.e., MZ2-MEL 3.1, MZ2-MEL 2.2, MZ2-MEL 2.2 ET1, NA8 MEL, and Mil3443.All of these lines are presented in Table 1, supra. Cells were cultured,and then lysed in a Nonidet P40 (NP-40) buffer (150 mM NaCl, 0.5% NP-40,2 mM EDTA, 80 mM Tris-HCl, pH 7.5, 0.02% NaN₃, 100 ug/ml PMSF and 100ug/ml aprotinin). Approximately 50 ug aliquots were then subjected toSDS-PAGE under reducing conditions, and the thus separated proteins weretransferred to nitrocellulose paper. Undiluted hybridoma supernatants,and a standard, commercially available electrochemiluminescencedetection system was used. FIG. 1 shows these results. The wereintriguing because two major bands were found by both mAbs when testingMZ2-MEL 3.1. These bands are at 46 and 72 kilodaltons. The known MAGE-1specific monoclonal antibody MA454 (Chen, et al., Proc. Natl. Acad. Sci.USA 91: 1004 -1008 (1994); U.S. Pat. No. 5,541,104)) did not detectanything in MAGE-1 negative cell line MZ2-MEL 2.2, but when this cellline was transfected with MAGE-1 cDNA (to become cell line MZ2-MEL 2.2ET1), MA 454 mAb did bind to a 46 kD band. one concludes from this thatthe 46 kilodalton species bound by all of MA454, 6C1, and 6F12, isMAGE-1 protein, but that the latter two mAbs are cross reactive with asecond, 72 kilodaltons protein which was expressed by MZ2-MEL 3.1,MZ2-MEL 2.2, and Mil3443 (as well as transfected MZ2 MEL 2.2. ET1).Note, however, that MZ2-MEL 2.2 is MAGE-1 negative, suggesting that thecross reactivity is with a non-MAGE-1 protein.

EXAMPLE 5

[0021] The fact that NA8-MEL did not express any of MAGE-1, 2, 3 or 4and did not produce any proteins which bound to any of the three mAbstested, made it useful in experiments to determine whether or notdetection of the 72 kDa protein was dependent on presence of MAGE-1. TheNA8-MEL cells were transiently transfected with MAGE-1 cDNA in plasmidpcDNAI, using lipofectin. The transfected cells were lysed, and analyzedvia western blotting, as described supra, using 6C1 and 6F12. A band of46 kilodaltons resulted, as did a faint band corresponding to what isbelieved to be a multimeric form of MAGE-1. See FIG. 2. No 72 kDa bandwas found, however. There was no 72 kDa protein found followingtransient transfection with each of MAGE-2, 3, 4 and 12. This was alsotrue with COS-7 cells, following transient transfection.

EXAMPLE 6

[0022] In view of the unexpected presence of the 72 kDalton band,Western blotting was carried out in accordance with the procedures setforth supra, on a large number of cells. The results are shown in Table2, which also presents results from MAGE-1, 2, 3 and 4 mRNA expressiontesting. There was no relationship observed between the 46 and 72kDalton proteins. TABLE II DETECTION OF MAGE-1 PROTEIN AND THE 72-kDaPROTEIN IN CELL LINES BY WESTERN BLOTTING WITH MAbs 6Cl AND 6F12 Westernblot MAGE-1 72-kDa MAGE-mRNA Cell lines protein protein expression^((a))MZ2 · MEL 3.1 + + 1^(⊕), 2^(⊕), 3^(⊕), 4^(⊕) ^((a))MZ2-MEL 2.2 − +1−, 2⁶¹ , 3⁶¹ , 4− ^((a))MZ2-MEL 2.2 ET1 + + 1^(⊕), 2^(⊕), 3^(⊕), 4−^((a))Mil3443 + + 1^(⊕), 2^(⊕), 3^(⊕), 4^(⊕) ^((a))NA8-MEL − − 1−, 2−,3−, 4−

(

)IGR39 − − 1−, 2−, 3−, 4− ^((a))Me272.L.N2 − − 1−, 2−, 3^(61 , 4−)^((a))Me220 − − 1−, 2−, 3−, 4− ^((a))IGR3 + + 1^(⊕), 2^(⊕), 3^(⊕), 4^(⊕)^((a))Me204.A.1 − − 1−, 2^(⊕), 3^(⊕), 4− ^((a))Me242.B.1 − − 1−, 2^(⊕),3−, 4−

(

)Me241.1 + + 1^(⊕), 2−, 3^(⊕), 4− ^((a))Me235 + + 1^(⊕), 2^(⊕), 3^(⊕),4− ^((a))Me192.2.20 + + 1^(⊕), 2^(⊕), 3^(⊕), 4^(⊕) ^((a))Mi9 − − 1−,2^(⊕), 3^(⊕), 4− ^((a))Mil3 − − 1−, 2^(⊕), 3^(⊕), 4− ^((a))Mi21 − − 1−,2−, 3^(⊕), 4− ^((a))Me24K.3 + + 1^(⊕), 2^(⊕), 3^(⊕), 4^(⊕)

(

)Me244.1 + + 1^(⊕), 2^(⊕), 3−, 4−

(

)Me222.6 + + 1^(⊕), 2^(⊕), 3^(⊕), 4− ^((a))M14 + + 1^(⊕), 2^(⊕), 3^(⊕),4^(⊕) ^((b))Cl-18 + − 1^(⊕), 2−, 3^(⊕), 4− ^((b))U251 − − 1−, 2−, 3−, 4−^((b))LN215 + + 1^(⊕), 2^(⊕), 3^(⊕), 4− ^((c))TF1 − − 1−, 2−, 3^(⊕), 4−^((c))HEL + − 1^(⊕), 2−, 3−, 4− ^((d))

− − 1−, 2−, 3−, 4− ^((e))ACN + − 1^(⊕), 2^(⊕), 3^(⊕), 4− ^((e))LAN-2 + +1^(⊕), 2^(⊕), 3^(⊕), 4^(⊕) ^((f))Fek4 − − 1−, 2−, 3−, 4−

EXAMPLE 7

[0023] It is known that MAGE-1 expression can be induced, in vitro, insome MAGE-1 mRNA negative cell lines, by 5-aza-2′-deoxycytidine, ahypomethylating agent (“DAC”). This agent was incubated with threeMAGE-1 mRNA negative cell lines (IGR 39, NA8-MEL, and U251), for 72hours, after which lysates were taken, and incubated with monoclonalantibody 6C1. This treatment induced production of both the 46 kDa andthe 72 kDa protein.

EXAMPLE 8

[0024] The intriguing results reported supra suggested furtherexperiments to determine the identify of the 72 kDa protein. First, amelanoma expression library was prepared from melanoma cell line MZ2-MEL43, using a commercially available system. Following the preparation,bacteriophages were plated (approximately 4×10⁵ pfus), and transferredto nitrocellulose filters. These were then blocked with 5% milk powderin phosphate buffered saline, and then incubated with monoclonalantibody 6C1 (hybridoma supernatant diluted 1:4 in RPMI/10% fetal calfserum). The materials were then washed with PBS/0.5% Tween-20 andincubated with horseradish peroxidase conjugated sheep anti-mouse IgG,diluted 1:3000 in PBS/5% milk powder in PBS. Another wash, with 5%Tween-20 followed. Signals were detected using ECL, as discussed supra.All positive plaques were subjected to secondary and tertiary screening.Positives were then picked and transferred to a tube containing phagelysis buffer (20 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.1% Tween 20), and analiquot of this (5 ul) was used to amplify phage inserts. These wereamplified via PCR, using:

[0025] GTGGCGACGA CTCCTGGAG (SEQ ID NO: 1)

[0026] and

[0027] CAGACCAACT GGTAATGGTA GCG (SEQ ID NO: 2)

[0028] which are λ primers. The cycling parameters were: 1 minute at 94°C., 1 minute at 61° C., and 1 minute at 72° C., for 30 cycles, followedby a final extension at 72° C., for 10 minutes.

[0029] A partial 5′ sequence of the clones was then obtained, using acommercially available sequencing kit, using SEQ ID NO: 1. See Casanova,Meth. Mol. Biol. 23: 191-197 (1993). Twelve clones were sequenced, andthree were found to be identical to that of the MAGE-10 genomicsequence, as reported by DePlaen et al., Immunogenetics 40: 360-369(1994), and Genbank Accession No. U10685.

[0030] One insert was then amplified, using SEQ ID NOS: 1 and 2, and acommercially available system. The cycling parameters for thisamplification were 15 seconds at 94° C., 30 seconds at 61° C., 1 minuteat 72° C. (10 cycles) , 15 seconds at 94° C., 30 seconds at 61° C., 80seconds plus 20 seconds cycle elongation at 72° C., for 20 cyclesfollowed by 10 minutes at 72° C., for a final extension. Theamplification product was cleaved with restriction endonucleases NotIand SalI, and then subcloned into Bluescript plasmid. Automatedsequencing was then carried out using T3 and T7 primers. It wasconfirmed to be a partial MAGE-10 cDNA sequence (1400 base pairs), whichcorresponded to a start at position 2770 at the 5′-end, and extending660 base pairs beyond the 3′-end of the genomic sequence reported byDePlaen, et al., supra.

EXAMPLE 9

[0031] Using the information obtained from the experiments described,supra, additional work was carried out to obtain a full length cDNAclone for MAGE-10.

[0032] As indicated, the partial cDNA clone was 1.4 kb long. Thisfragment was subjected to digestion with restriction endonucleases, andan HpaI fragment, corresponding to nucleotides 2770-3510 of the known,gDNA sequence, was isolated, and ³²p labeled, using a random priming DNAlabeling kit. The labeled probe was then used to screen two librariesfrom a melanoma cell line (Lyse-4), in the vectors pcDNAI/Amp and pCEP4.The hybridization was carried out on filters, using 5×SSC, 5×Denhardt's,0.5% SDS, and 100 ug/ml denatured salmon sperm DNA, at 65° C. Filterswere then washed three times for 10 minutes at room temperature, with1×SSC, 0.1% SDS, once for 20 minutes at 65° C., with 1×SSC, 0.1% SDS,and twice for 20 minutes at 65° C., with 0.1×SSC, 0.1% SDS. Ten positiveclones were found, and sequenced automatically, using T7 and SP6 primersfor the pcDNAI/Amp vector, and the pCEP-4 forward primer for pCEP-4.Several MAGE-10 clones were isolated, and fell into two categories (2.5kb, and 1.5 kb, respectively), with different 3′-ends. The differencemay result from alternate oligo (dT) priming during the cDNA synthesisfor the library. The clones all seemed to be identical but for the first50-70 nucleotides at the 5′-end. Comparison to the known, genomicsequence delineated existence of at least four exons, the last two beingidentical to those predicted by DePlaen, et al. supra (positions1740-1814, and 1890-end). The second exon corresponded to positions603-701, while the first exon did not appear to correspond to anypreviously recognized MAGE-10 sequence. The open reading frame was foundin the last exon. A sequence is set forth at SEQ ID NO: 3. The first 100bases or so indicate consensus sequences, based upon the collectivesequence information secured via these experiments.

EXAMPLE 10

[0033] Three clones were isolated from the pcDNAI/Amp library, describedsupra, and were used for in vitro transcription and translation. Theseinserts were about 1.5 kilobases long, terminating at about position3156, using genomic sequence enumeration. One ug of each DNA wastranslated, using a commercially available system, and a luciferasecontrol plasmid was used as control. Translation products were subjectedto PAGE analysis, and duplicate gels of non-radioactively labeledproduct were transferred to membranes, where Western Blotting wascarried out, using mAb 6C1, or polyclonal antibodies prepared asdescribed infra. Radiolabelled materials showed a 72 kilodalton proteinfrom all three clones tested, suggesting that the mAb was cross reactivewith MAGE-1 and MAGE-10.

EXAMPLE 11

[0034] Polyclonal antiserum against MAGE-10 was made as follows.

[0035] Immunogenic, MAGE-10 derived peptide

[0036] (H) QDRIATTDDTTAMASASSSATGSFSYPE (OH)

[0037] (SEQ ID NO: 4),

[0038] a portion of the deduced amino acid sequence of MAGE-10 was made,as were hybrids of this peptide and helper peptide P-30.

[0039] Helper Peptide P30 is well known, as per Valmori, et al., J.Immunol. 149: 717-721 (1992). It is a tetanus toxin T cell epitope, withamino acid sequence:

[0040] FNNFTVSFWLRVPKVSASHLE

[0041] (SEQ ID NO: 5). Peptides were dissolved at 400 ug/ml in 100 mMTris-HCl, pH 7.5, 0.9% NaCl. A rabbit was immunized over a 56 dayperiod, with hybrid peptide (0.5 ml) at day O, the MAGE-10 peptide (0.5ml) at day 14, a second 0.5 ml injection of hybrid at day 28, and afinal injection at day 56, of 0.5 ml of the MAGE-10 peptide. Antiserumproduced in accordance with this protocol was tested for reactivity withMAGE-10 in various assays. Specifically, the in vitro translationproduct of expression of cDNA corresponding to SEQ ID NO: 3 was testedin Western blotting experiments, along the lines set forth supra. Theantiserum was found to bind to a protein which was produced via the invitro expression. It also recognized a 72 kDa band from melanomalysates. In an ELISA, the polyclonal antibodies were found to recognizethe MAGE-10 peptide.

[0042] The foregoing experiments describe the production of monoclonalantibodies which specifically bind to a tumor rejection antigenprecursor TRAP.

[0043] The invention thus relates to MAGE-10 binding monoclonalantibodies and the hybridomas which produce them. The mAbs wee found tobe useful in determining expression of MAGE-10. The mAbs can be added,e.g., in labeled form, bound to a solid phase, or otherwise treated toincrease the sensitivity of MAGE-10 detection. Any of the standard typesof immunoassays, including ELISAs, RIAs, competitive assays,agglutination assays, and all others are encompassed with respect to theway the mAbs can be used. The detection of MAGE-10 expression product isuseful, e.g., in diagnosing or monitoring the presence or progression ofa cancer.

[0044] The isolated, MAGE-10 protein is also a feature of thisinvention. This molecule has a molecular weight of about 72 kDa asdetermined by SDS-PAGE, and is useful as an immunogen as is the peptideof SEQ ID NO: 4, shown by the examples to be immunogenic. Preferably,these are used in combination with a suitable adjuvant.

[0045] Isolated cDNA encoding MAGE-10 is also a feature of thisinvention, such as the cDNA of SEQ ID NO: 3. Also a part of theinvention are cDNA molecules which have complementary sequences thathybridizes to SEQ ID NO: 3 under stringent conditions (e.g., 0.2×SSC,0.1% SDS at 65° C. or, more preferably, 0.1×SSC). These should include,as a minimum, nucleotides 164-574 of SEQ ID NO: 3, in 5′ to 3′ order.Nucleic acid molecules consisting of nucleotides 164-185, and 553-574 ofSEQ ID NO: 3 are especially useful as probes and/or primers, and arealso a part of this invention. The sequences can be used in the form ofexpression vectors when operably linked to promoters, and then used totransform or transfect cells, to produce various recombinant eukaryoticcell lines and prokaryotic cell strains. Similarly, the sequences, andsequences such as SEQ ID NOS: 1 and 2 can be used in varioushybridization assays, such as PCR based assays. These are well known tothe skilled artisan, and need not be repeated here.

[0046] The terms and expression which have been employed are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expression of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

1 5 19 nucleotides nucleic acid single linear 1 GTGGCGACGA CTCCTGGAG 1923 nucleotides nucleic acid single linear 2 CAGACCAACT GGTAATGATA GCG 232559 nucleotides nucleic acid single linear 3 TCCGGGGTCG CTCGAGCCGGCCGGGACTCG GGGATCASAA GTAACGGCGG 50 YYMKYGTKCT GAGGGACAGG CTTGAGATCGGCTGAAGAGA GCGGGCCCAG 100 GCTCTGTGAG GAGGCAAGGG AGGTGAGAAC CTTGCTCTCAGAGGGTGACT 150 CAAGTCAACA CAGGGAACCC CTCTTTTCTA CAGACACAGT GGGTCGCAGG200 ATCTGACAAG AGTCCAGGTT CTCAGGGGAC AGGGAGAGCA AGAGGTCAAG 250AGCTGTGGGA CACCACAGAG CAGCACTGAA GGAGAAGACC TGCCTGTGGG 300 TCCCCATCGCCCAAGTCCTG CCCACACTCC CACCTGCTAC CCTGATCAGA 350 GTCATCATGC CTCGAGCTCCAAAGCGTCAG CGCTGCATGC CTGAAGAAGA 400 TCTTCAATCC CAAAGTGAGA CACAGGGCCTCGAGGGTGCA CAGGCTCCCC 450 TGGCTGTGGA GGAGGATGCT TCATCATCCA CTTCCACCAGCTCCTCTTTT 500 CCATCCTCTT TTCCCTCCTC CTCCTCTTCC TCCTCCTCCT CCTGCTATCC550 TCTAATACCA AGCACCCCAG AGGAGGTTTC TGCTGATGAT GAGACACCAA 600ATCCTCCCCA GAGTGCTCAG ATAGCCTGCT CCTCCCCCTC GGTCGTTGCT 650 TCCCTTCCATTAGATCAATC TGATGAGGGC TCCAGCAGCC AAAAGGAGGA 700 GAGTCCAAGC ACCCTACAGGTCCTGCCAGA CAGTGAGTCT TTACCCAGAA 750 GTGAGATAGA TGAAAAGGTG ACTGATTTGGTGCAGTTTCT GCTCTTCAAG 800 TATCAAATGA AGGAGCCGAT CACAAAGGCA GAAATACTGGAGAGTGTCAT 850 AAAAAATTAT GAAGACCACT TCCCTTTGTT GTTTAGTGAA GCCTCCGAGT900 GCATGCTGCT GGTCTTTGGC ATTGATGTAA AGGAAGTGGA TCCCACTGGC 950CACTCCTTTG TCCTTGTCAC CTCCCTGGGC CTCACCTATG ATGGGATGCT 1000 GAGTGATGTCCAGAGCATGC CCAAGACTGG CATTCTCATA CTTATCCTAA 1050 GCATAATCTT CATAGAGGGCTACTGCACCC CTGAGGAGGT CATCTGGGAA 1100 GCACTGAATA TGATGGGGCT GTATGATGGGATGGAGCACC TCATTTATGG 1150 GGAGCCCAGG AAGCTGCTCA CCCAAGATTG GGTGCAGGAAAACTACCTGG 1200 AGTACCGGCA GGTGCCTGGC AGTGATCCTG CACGGTATGA GTTTCTGTGG1250 GGTCCAAGGG CTCATGCTGA AATTAGGAAG ATGAGTCTCC TGAAATTTTT 1300GGCCAAGGTA AATGGGAGTG ATCCAAGATC CTTCCCACTG TGGTATGAGG 1350 AGGCTTTGAAAGATGAGGAA GAGAGAGCCC AGGACAGAAT TGCCACCACA 1400 GATGATACTA CTGCCATGGCCAGTGCAAGT TCTAGCGCTA CAGGTAGCTT 1450 CTCCTACCCT GAATAAAGTA AGACAGATTCTTCACTGTGT TTTAAAAGGC 1500 AAGTCAAATA CCACATGATT TTACTCATAT GTGGAATCTAAAAAAAAAAA 1550 AAAAAAAAGT TGGTATCATG GAAGTAGAGA GTAGAGCAGT AGTTACATTA1600 CAATTAAATA GGAGGAATAA GTTCTAGTGT TCTATTGCAC AGTAGGATGA 1650CTATAGTTAA CATTAAGATA TTGTATATTA CAAAACAGCT AGAAGGAAGG 1700 CTTTTCAATATTGTCACCAA AAAGAAATGA TAAATGCATG AGGTGATGGA 1750 TACACTACCT GATGTGATCATTATACTACA TATACATGAA TCAGAACATC 1800 AAATTGTACC TCATAAATAT CTACAATTACATGTCAGTTT TTGTTTATGT 1850 TTTTGTTTTT TTTTAATTTA TGAAAACAAA TGAGAATGGAAATCAATGAT 1900 GTATGTGGTG GAGGGCCAGG CTGAGGCTGA GGAAAATACA GTGCATAACA1950 TCTTTGTCTT ACTGTTTTCT TTGGATAACC TGGGGACTTC TTTTCTTTTC 2000TTCTTGGTAT TTTATTTTCT TTTTCTTCTT CTTCTTTTTT TTTTTTAACA 2050 AAGTCTCACTCTATTGCTCT GGCAGGAGTG CAGTGGTGCA GTCTCGGCTC 2100 ACTGCAACTT CCGCCTCCTGGGTTCAAGCG ATTCTCCTGC CTCAGTCTCC 2150 TGAGTAGCTG GGATTACAAG TGTGCACCACCATACCCGGC TAATTTTGTA 2200 TTTTTTAGTA GAGATGGGGT TTCACCATGT TGGCCAGGCTGGTCTCAAAC 2250 TCCTGACCTC AGGTAATCTG CCCGCCTCAG CCTCCCAAAG TGCTGGGATA2300 ACAGGTGTGA GCCCACTGCA CCCCAGCCTC TTCTTGGTAT TTTAAAATGT 2350TGTTACTTTT ACTAGAATGT TTATGAGCTT CAGAATCTAA GGTCACACGT 2400 TCGTTTCTGTTTATCCAGTT TAAGAAACAG TTTTGCTATT TTGTAAAACA 2450 AATTGGGAAC CCTTCCATCATATTTGTAAT CTTTAATAAA ATAACATGGA 2500 ATTGGAATAG TAATTTTCTT GGAAATATGAAAAAATAGTA AAATAGAGAA 2550 AATAATTTT 2559 28 amino acid linear 4 Gln AspArg Ile Ala Thr Thr Asp Asp Thr Thr Ala Met Ala Ser Ala 5 10 15 Ser SerSer Ala Thr Gly Ser Phe Ser Tyr Pro Glu 20 25 21 amino acid linear 5 PheAsn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser 5 10 15 AlaSer His Leu Glu 20

We claim:
 1. Isolated cDNA molecule which encodes MAGE-10 tumorrejection antigen precursor, the complementary sequence of whichhybridizes to nucleotides 164-574 SEQ ID NO: 3, under stringentconditions.
 2. The isolated cDNA molecule of claim 1, comprisingnucleotides 67-2559 of SEQ ID NO:
 3. 3. The isolated cDNA molecule ofclaim 1, comprising SEQ ID NO:
 3. 4. Expression vector comprising theisolated cDNA molecule of claim 1, operably linked to a promoter. 5.Eukaryotic cell line or prokaryotic cell strain, transfected ortransformed with the expression of claim
 3. 6. Isolated MAGE-10 tumorrejection antigen precursor having a molecular weight of about 72kilodaltons as determined by SDS-PAGE.
 7. Isolated MAGE-10 tumorrejection antigen precursor encoded by the isolated cDNA molecule ofclaim
 1. 8. Monoclonal antibody which binds to the isolated tumorrejection antigen precursor of claim
 6. 9. Isolated tumor rejectionantigen precursor derived peptide consisting of the amino acid sequenceof SEQ ID NO:
 4. 10. Immunogenic composition comprising the isolatedMAGE-10 tumor rejection antigen precursor of claim 6, and an adjuvant.11. Immunogenic composition comprising the peptide of claim 9 and anadjuvant.
 12. Polyclonal antiserum obtained by immunizing a non-humananimal with the peptide of claim 8 under conditions favoring an immuneresponse thereto, and isolating antiserum produced thereby.
 13. Methodfor determining presence of MAGE-10 tumor rejection antigen precursor ina sample, comprising contacting said sample with the monoclonal antibodyof claim 8 and determining binding therebetween.
 14. Method fordetermining presence of MAGE-10 tumor rejection antigen precursor in asample comprising contacting said sample with the polyclonal antiserumof claim 12, and determining binding therebetween.
 15. Hybridoma cell inwhich produces the monoclonal antibody of claim
 8. 16. The monoclonalantibody of claim 8, wherein said antibody is humanized.